Materials exhibiting biomimetic carbon fixation and self-repair

ABSTRACT

A composition can photocatalytically reduce carbon dioxide.

CLAIM OF PRIORITY

This application claims priority to U.S. Provisional Patent No.62/740,376, filed Oct. 2, 2018, which is incorporated by reference inits entirety.

GOVERNMENT SPONSORSHIP STATEMENT

This invention was made with Government support under Grant No.DE-FG02-08ER46488 awarded by the Department of Energy (DOE). TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to materials capable of carbon dioxide fixation.

BACKGROUND

Carbon dioxide is the main source for production of many chemicalsincluding methanol and methane. Strategies for CO₂ reduction yieldvarious products with different yield and selectivity. However, theseapproaches share the high energy consumption rates, using valuablereactants such as H₂, and possibly emitting more net CO₂ to atmosphere.

SUMMARY

In one aspect, a method of sequestering carbon dioxide can includeexposing a composition including a catalyst to carbon dioxide, andreducing the carbon dioxide with the catalyst with light energy,chemical energy or electrical energy to form formaldehyde or aformaldehyde product.

In another aspect, a method of self-healing a polymer matrix can includeexposing a polymer matrix including a catalyst to carbon dioxide and anenergy source and generating additional material to the polymer matrixfrom the carbon dioxide.

In another aspect, a composition can include a polymer matrix includinga catalyst configured to generate additional material to the polymermatrix from carbon dioxide with light energy, chemical energy orelectrical energy to form formaldehyde or a formaldehyde product.

In certain circumstances, the catalyst can include a chloroplast, ananocatalyst, or a colloidal battery. For example, the composition caninclude a chloroplast in a hydrogel.

In certain circumstances, the composition can include a nanoparticle,for example, particles can have a size of 2 nm to 500 nm. Thenanoparticle can include a metal oxide or metal sulfide, for example,titania or ceria.

In certain circumstances, the composition can include a polymer matrix.

In certain circumstances, the composition can include an enzyme. Forexample, the composition can include a glucosidase, a glucosedehydrogenase or a hexokinase.

In certain circumstances, the composition can include a substrate. Forexample, the substrate can be a graphene oxide.

In certain circumstances, the composition can include a monomer. Forexample, the monomer can include a styrene, an acrylate or anacrylamide.

In certain circumstances, the formaldehyde product can include a ureaformaldehyde polymer, a trimethylene oxide, or polyoxymethylene.

Other aspects, embodiments, and features will be apparent from thefollowing description, the drawings, and the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B depict a schematic illustration of a synthetic material thatgrows, strengthens and self-repairs with embedded plant chloroplasts. InFIG. 1A, GPMAA forms hydrogel as lightly cross-linked by hydrogenbonding in water. The hydrogel continuously grows, strengthens andself-repairs as long as chloroplasts carry out carbon fixation andexport glucose. In FIG. 1B, chloroplasts transform solar energy andcarbon dioxide into the chemical energy and the assimilated carbon inthe form of triose phosphate during the day. Alternatively, chloroplastsexport maltose and glucose resulting from the breakdown of starchthrough the translocators at night. Exported glucose and glucose fromenzymatic hydrolysis of maltose are converted to gluconolactone (GL) byGOx, and subsequently react to primary amine functionalizedmethacrylamide (APMA) and polymerize to glucose-containingpolymethacryamide (GPMAA) in the medium.

FIGS. 2A-2D depict in vitro a carbon fixing hydrogel formation fromgluconolactone and 3-aminopropyl methacrylamide. FIG. 2A shows FT-IRspectra of the mixture of GL and APMA at 0 h, GPMAA and GO-GPMAA in 5 hunder the light. FIG. 2B shows rheology properties of GPMAA (squares)and graphene oxide (GO) containing GPMAA (triangles) at differentreaction time. The data are expressed as the average±SD (n=3). FIG. 2Cshows a schematic illustration on separation of GPMAA hydrogel. FIG. 2Dshows formation of fibrillar structure during the separation process.Two separated thin GPMAA hydrogels are formed on the glass slides andthen they are attached to each other. The glass slides are pulledseparating the hydrogels at a rate of 0.04 mm/sec.

FIGS. 3A-3F depict a systematic investigation on boosting glucosesynthesis and export from isolated chloroplast. FIG. 3A shows glucoseconcentration with and without maltose hydrolysis by a-glucosidase (24 UmL−1) at light and dark period. Total incubation time is 8 h with 4 h oflight and 4 h of dark. FIG. 3B shows glucose concentration in differentillumination time; 12 h light and 12 h dark (black squares), continuousillumination for 24 h (blue circles), and 12 h light and 12 h dark with5 mM Pi supply at the beginning of dark period (pink triangles). FIG. 3Cshows an effect of chloroplast proton gradient or ATP on glucose export.Chloroplast medium is adjusted from optimum pH 7.6 (control, blacksquares) to pH 8.0 (pink triangles) to induce alkalinization ofchloroplast stroma, and 2 mM ATP is added to the medium (blue circles)to enhance active transport mechanism of glucose. FIG. 3D shows acomparison of approaches to boost glucose export under presence orabsence of external Pi supply. A mixture of hexokinase (10 U mL−1), 0.6mM ATP, with 5 mM Pi (blue circles) or without additional Pi (pinktriangles), or 5 mM Pi alone (black squares) is added to the chloroplastsuspension medium at every hour during the dark period. Black arrowsindicate the addition of 5 mM Pi. FIG. 3E shows an effect of nanoceriaon glucose export after 12 h light period. Chloroplasts are incubated inpresence of nanoceria 3 h prior to the light period. Final concentrationof nanoceria is 5 μM (blue circles) or 50 μM (pink triangles). Controlmeans chloroplast incubated for the experimental period withoutnanoceria (black squares). Green arrows indicate the addition ofhexokinase reaction mixture. White or black rectangles on the top ofeach graph imply light or dark period, respectively. FIG. 3F shows GLconcentration from chloroplasts incubated in the medium containing 20 UmL−1 GOx under the continuous illumination for 5 h. White or blackrectangles on the top of each graph imply light or dark period,respectively. GL concentration is presented by gluconic acid afterhydrolysis in 2M NaOH. The data are expressed as the average±S.D.(n=5-10). *P, **P<0.05 compared with control [Glucose] at 0 h.

FIGS. 4A-4D depict hydrogel growth over time from ambient carbon dioxideand light around isolated chloroplasts. FIG. 4A shows a microscopeimages of growing hydrogel near the isolated chloroplasts in the mediumcontaining GOx (20 U mL−1) and 0.1% w/v APMA. Exposure conditions areambient CO₂ and 18 h ambient illumination after 1 h dark period. Scalebars are 5 μm. FIG. 4B shows a schematic illustration of hydrogelformation on the graphene oxide film. Isolated chloroplasts areincubated with GOx immobilized graphene oxide film in the mediumcontaining 0.1% APMA. Followed by glucose export from chloroplasts,glucose is converted into GL on the graphene oxide surface by GOxfollowed by reacting to APMA, which polymerizes to form GPMAA. FIG. 4Cshows a characteristic Raman bands of GPMAA hydrogel. FIG. 4D shows anoptical image of chloroplast embedded-GPMAA hydrogel (left) and Ramanmapping (right) based on the characteristic Raman bands ratio between1245 and 1290 cm⁻¹ under a laser excitation of 632 nm. Scale bars are 2μm.

FIGS. 5A-5D depict self-repair property of GPMAA hydrogel. FIG. 5A showspartially repaired hydrogels by exposure to light overnight, hydrogelsare dyed yellow and blue to allow for easily distinguished interface.FIG. 5B shows fully repaired hydrogels by addition of 1 M GL (5 μL) tothe interface and exposure to light overnight. FIG. 5C shows shearstrength restoration of hydrogels by physical attachment for 30 min(blue line) or exposure to light overnight after addition of GL (redline). Two separated thin GPMAA hydrogels are formed on the glass slidesand then they are attached to each other. The glass slides are pulledseparating the hydrogels at a rate of 0.04 mm/sec. FIG. 5D shows aschematic illustration of self-healing mechanism of chloroplast embeddedhydrogel matrix. Glucose molecules supplied by chloroplasts repair thelocal damage by exceeding its own local material balance through theatmospheric CO₂ fixation.

FIG. 6A-6E depict FT-IR spectra of buffer, AMPA, and GL. Thecharacteristic peak of new amide bond between AMPA and GL is notobserved.

FIG. 7 depicts FT-IR spectra of GPMAA with different ratio of GL andAPMA. Panel A shows 1 equiv. of APMA reacts to GL, and panel B shows 5equiv. of APMA reacts to GL. GL is partially hydrolyzed to gluconic acidin water by equilibrium, which will instead form a charged complex withamine group of APMA. The rate of hydrolysis of GL is faster at basic pHand high temperature. Therefore, the reaction condition has to becarefully adjusted. When 1 equiv. of APMA reacts to GL, unreacted GLstill remains in a form of gluconic acid, which indicates the excessAPMA needs to increase yield of GPMAA. For example, the peak of gluconicacid C═O at 1734.5 cm⁻¹ disappears in the hydrogel by reacting it withexcess APMA (5 equiv.).

FIGS. 8A-8D depict rheology properties of GPMAA hydrogel. FIGS. 8A-Cshow different time intervals, (FIG. 8A) 2 h, (FIG. 8B) 6 h, and (FIG.8C) 18 h. Frequency sweep on GPMAA hydrogel shows a more solid-likebehavior (G′>G″). FIG. 8D shows corresponding recovery of GPMAA hydrogelunder shear stress (strain 100%, black) and after removal of shearstress (strain 1% black).

FIGS. 9A-9D show rheology property of graphene oxide-GPMAA hydrogel.FIGS. 9A-9C show different time intervals, (FIG. 9A) 2 h, (FIG. 9B) 6 h,and (FIG. 9C) 18 h. Frequency sweep on GPMAA hydrogel shows a moresolid-like behavior (G′>G″). FIG. 9D shows corresponding recovery ofGPMAA hydrogel under shear stress (strain 100%, black) and after removalof shear stress (strain 1% black).

FIGS. 10A-10C depict indentation curves. FIG. 10A shows a representativeindentation curve for the swollen GPMAA gel with 50 wt % water content.FIG. 10B shows a representative indentation curve for the dry GPMAA.FIG. 10C shows Young's moduli measured with both sharp and colloidalprobes (n=5). It is known that sharp probes result in smalloverestimation because the contact surface area is difficult to beprecisely determined.

FIGS. 11A-11C depict measurement of glucose exported from isolatedchloroplasts on the microfluidic chip. FIG. 11A shows a schematic layoutshowing a microfluidic chip with a chamber for chloroplasts (greenellipsoids) and microsieves to extract produced glucose (small redspheroids). FIG. 11B shows a photo of the microfluidic chip. Scale baris 1 cm. FIG. 11C shows glucose concentration with or without nanoceria.

FIG. 12 depicts glucose export in presence of APMA. Relative glucoseconcentration in the chloroplasts medium containing 0% or 0.1% or 0.4%w/v APMA. The data are expressed as the average±SD (n=3).

FIG. 13 depicts an FT-IR spectrum of chloroplast embedded GPMAAhydrogel. Isolated chloroplasts were kept under the ambient lightovernight. The chloroplast medium contained GOx (20 U mL−1) and 0.1%APMA. IR peaks were confirmed by Fourier transform infrared spectroscopy(FT-IR) spectroscopy with an FTIR microscope.

FIG. 14 depicts Raman spectra of chloroplast buffer alone (blue), thebuffer containing 20 U/mL glucose oxidase (red) or 0.1% (w/v) APMA(green).

FIG. 15 depicts a comparison of Raman spectra between APMA and GPMAA.

FIGS. 16A-16B depict Raman spectra of chloroplast suspension containingglucose oxidase and AMPA. Isolated chloroplasts are incubated in thepresence of glucose oxidase (20 U/mL) and 0.1% (w/v) AMPA overnight inthe dark (FIG. 16A) or under the light (FIG. 16B). The stacked Ramanspectra are obtained from multiple spots near the chloroplasts.

FIG. 17 depicts deformation of incomplete-self repaired hydrogel. GPMAAare dyed blue and yellow to allow for easily distinguished interface.The physically attached two hydrogels, which are kept under the darkovernight, are easily separated by deformation.

FIG. 18 depicts a schematic Illustration of System II: (1) CO₂photocatalytic reduction to formaldehyde, (2) Formaldehyde trimerizationto trioxane, and (3) Trioxane polymerization to polyoxymethylene (POM).

FIG. 19 depicts a proposed kinetic model for CO₂ System II catalyticphotoreduction to HCOH and then to polyoxymethylene (POM).

FIG. 20 depicts a time profile of the products in the catalyticphotoreduction of CO₂: the kinetic model fitted versus experimental dataof catalytic pathway products.

FIG. 21 depicts a time profile of the production of 1,3,5-trioxane inCompartment 2 of the proposed system from formaldehyde produced incompartment 1 by photocatalytic reduction of CO₂.

FIG. 22 depicts a schematic of the proposed transport and polymerizationof glucose. Panel A shows transfer function model of sugar export of theChloroplast. Panel B shows Intra-matrix transport and polymerizationschematic

FIG. 23 depicts the novel carbon-fixing polyoxymethylene composite maybe realized by an overall pathway featuring a compartmental catalyticsystem consisted of: (panel a) photocatalyst; monomer formationcatalyst; and polymerization initiator exposed to atmospheric CO₂ andsunlight. Panel b shows the full chemical pathway consists of threesteps: RXN 1: CO₂ photocatalytic reduction to formaldehyde, RXN 2:Formaldehyde conversion to 1,3,5-trioxane, RXN 3: Trioxanepolymerization to POM.

FIGS. 24A-24C depict kinetic experimental data and fitted model data for(FIG. 24A) RXN1: photocatalytic reduction of CO₂ to formaldehyde(experimental data obtained from Liu et al), (FIG. 24B) RXN2: Conversionof formaldehyde to trioxane (experimental data obtained from Yin et al),and (FIG. 24C) RXN3: Trioxane polymerization to polyoxymethylene (POM)(experimental data obtained from Shieh et al).

FIG. 25 depicts the proposed overall reaction network from CO₂ to:kinetic engineering is required to overcome two main bottlenecks in theprocess: (i) enhancing photocatalytic activity to improve the slow CO₂reduction reaction (RXN1, the green section of the network), and (ii)engineering trioxane formation and polymerization to dominate thecompeting pathway toward methanol and methane (RXN2 and RXN3, bluesection of the network).

FIG. 26A depicts the effect of relative improvement in kinetics of RXN 1(photocatalytic unit) and RXN 2&3 (formaldehyde polymerization unit) onPOM growth rate over time under atmospheric CO₂ pressure. At any givenCO₂ concentration, engineering of formaldehyde polymerization reactionunits accelerates overall kinetics until it reaches the POM steady stategrowth rate. Above this point, the overall process can be improved onlyby enhancing the photocatalytic activity in RXN 1. FIG. 26B depicts theeffect of relative improvement in kinetics of reaction network atvarious CO₂ adsorption capacities: CO₂ inflection concentrationseparates two regimes: (i) below CO_(2_inf) formaldehyde polymerizationis rate-limiting step and must be improved, (ii) above CO_(2_inf) POMgrowth rate linearly depends on the photocatalytic activity and CO₂concentration at the photocatalyst surface.

FIGS. 27A-27B depict the mapping of CO_(2_inf) with respect to theenhancement required for (FIG. 27A) RXN2 vs. RXN3 and (FIG. 27B) RXN1vs. RXN2 & 3. Depending on the reaction condition, we can determine thespecific focus of debottlenecking efforts, either polymerizationengineering and photocatalytic improvement or CO2 capturing viaadsorption.

DETAILED DESCRIPTION

In one example, one can learn from the mechanisms of self-assembly andself-repair displayed particularly in living plant systems to createhuman-synthesized analogs that benefit from these higher functionsoperating under non-biological conditions. Here, recent efforts inengineering biomimetic systems that exploit ambient solar energyharvesting and carbon dioxide conversion to high-energy products such asglucose and its polymeric derivatives are highlighted. By performingthese reactions compartmentally, it is possible to create materials thatgrow and self-repair using carbon dioxide as a carbon source. Suchmaterials would significantly benefit transportation and constructcosts, as well as exhibit self-healing and densification over time. Twosystems are detailed below.

The first involves the extraction of functional plant chloroplasts frombiomass and using them as embedded, functional photocatalysts for theproduction of glucose and starches from ambient solar energy andatmospheric carbon dioxide. Glucose can be converted to gluconolactone(GL) by glucose oxidase (GOx), which can then readily react withnucleophiles, such as the primary amine group (—NH₃) to generate agrowing polymer matrix. The importance of inorganic phosphate (Pi)concentration, glucose equilibrium across the chloroplast membrane, andthe concentration of photo-generated reactive oxygen species (ROS)towards glucose export efficiency have been investigated. Glucose exportfrom the isolated chloroplasts to gain quantifiable molecules forbuilding of a self-growing material was enhanced. Isolated chloroplastsare placed on the GOx immobilized-graphene oxide film in buffercontaining co-monomer, 6-aminopropyl methacrylamide (APMA). In thepresence of ambient light and exposure to atmospheric carbon dioxide for18 hours at room temperature, the formation of hydrogel-like materialwas observed around the chloroplast membrane as confirmed by Ramanspectroscopy 3D mapping. These efforts have benefited from a newtechnique, Lipid Exchange Envelope Penetration (LEEP) developed at MITfor the incorporation of nanoparticles into living plants, protoplastsand chloroplasts in vivo. This method allows for the incorporation ofchemo-protective, stabilizing and photoactive nanoparticles into thechloroplast to preserve and extend its catalytic function.

In one aspect, a method of sequestering carbon dioxide can includeexposing a composition including a catalyst to carbon dioxide, andreducing the carbon dioxide with the catalyst with light energy,chemical energy or electrical energy to form formaldehyde or aformaldehyde product.

In another aspect, a method of self-healing a polymer matrix can includeexposing a polymer matrix including a catalyst to carbon dioxide and anenergy source and generating additional material to the polymer matrixfrom the carbon dioxide.

In another aspect, a composition can include a polymer matrix includinga catalyst configured to generate additional material to the polymermatrix from carbon dioxide with light energy, chemical energy orelectrical energy to form formaldehyde or a formaldehyde product.

The enzymes or other additives can remove photo-generated reactiveoxygen species inside chloroplasts can extend the lifetime of isolatedchloroplasts, which ultimately translates into higher glucoseaccumulation in the medium. In another example, glucose export increasesonly after the addition of hexokinase, which acts as a sink for thisflux outside of the chloroplast.

Embedded, extracted chloroplasts can be carbon-fixing photocatalysts,which utilize abundant atmospheric carbon dioxide and solar energy toproduce reduction products. The material that can autonomously grow,strengthen and repair itself in response to certain types of damage. Forexample, separated hydrogels are able to seamlessly recombine upon lightexposure (FIGS. 5A-5D).

In certain circumstances, the catalyst can include a chloroplast, ananocatalyst, or a colloidal battery. For example, the composition caninclude a chloroplast in a hydrogel.

In certain circumstances, the composition can include a nanoparticle,for example, particles can have a size of 2 nm to 500 nm. Thenanoparticle can include a metal oxide or metal sulfide, for example,titania or ceria.

In certain circumstances, the composition can include a polymer matrix.

In certain circumstances, the composition can include an enzyme. Forexample, the composition can include a glucosidase (α-glucosidase), aglucose dehydrogenase or a hexokinase.

In certain circumstances, the composition can include a substrate. Forexample, the substrate can be a graphene oxide.

In certain circumstances, the composition can include a monomer. Forexample, the monomer can include a styrene, an acrylate or anacrylamide.

In certain circumstances, the formaldehyde product can include a ureaformaldehyde polymer, a trimethylene oxide, or polyoxymethylene. Thecomposition generates formaldehyde, thus, under reaction with urea,formaldehyde can produce Urea formaldehyde (UF), also known asurea-methanal.

As a next generation material system, the function of the chloroplastwith a semiconducting photocatalyst such as TiO₂ or graphitic C₃N₄ fordirect CO₂ reduction to formaldehyde was replaced. Domains performingthis chemistry under ambient conditions can be coupled into a materialwith differing pH to generate 1,3,5-trioxane and polymerize to linearpolyoxymethylene with a boron trifluoride (BF₃) or boron trifluoridediethyl etherate (BF₃OEt₂) as initiator. This system uses atmosphericCO₂ and converts it rapidly, avoiding the problem of intermediatestorage of the carbon from CO₂ and the associated energy expenditure.The final product is a lightweight, portable polymeric structure thatcan react with atmospheric CO₂, densify, and self-repair in the presenceof sunlight. Synthetic efforts going forward examine hierarchicalintegration and self-healing of both systems, coupled to a theoreticalframework within which the design and function of these fundamentallynew types of materials is explored. The mechanistic details of Systems Iand II are described below.

System 1: Materials with Embedded, Functional Plant Chloroplasts asPhotocatalysts for Glucose to Monomer to Polymer Matrix Production fromAmbient Solar Energy and CO₂

Materials capable of dynamic self-repair are commonly found among livingscaffolds and tissues. Correcting damage through self-repair mechanismspromise enhanced material lifetimes and increased resistance fromfatigue and acute mechanical stress. There has been a concerted researcheffort to develop synthetic materials mimicking aspects this naturalproperty by dynamic chemistry based on either covalent bonds ornon-covalent interactions that form or break reversibly. However, animportant distinction can be made here. These dynamic chemicalapproaches necessarily require one or more external stimuli such asheating, pH, mechanical stress, UV light, and external chemicaltreatment. Alternatively, autonomous systems, defined as materials thatthemselves can detect and respond to damage, have been recentlyintroduced. An encapsulated-monomer approach was first reported by Whiteand co-workers in 2001, in which reservoirs of a monomer and apolymerization initiator or catalyst are contained within the bulk ofthe material. A variant approach using bacteria (Bacillussphaericus)-induced carbonate precipitation was utilized formicro-cracks healing on a concrete material, but bacteria action waslimited due to lack of the local energy sources and low survivability inconcrete. The energy source limitation may in-fact translate into afundamental one for the use of heterotrophic living organisms inmaterials.

Herein, a new direction in self-healing materials as combining theautonomy of damage response with the ability to exceed the material'sown local material balance has been identified. To this end, a novelclass of material designed to grow, repair and strengthen through carbonfixation was created. By using embedded, extracted chloroplasts ascarbon-fixing photocatalysts, utilize abundant atmospheric CO₂ and solarenergy (FIGS. 1A-1B) as drivers. The chemistry of material synthesis touse glucose, a saccharide exported from chloroplasts, for facilereaction under relatively mild conditions was designed. Severalstrategies are systematically investigated to improve glucose exportfrom isolated chloroplasts, shown to be the limiting factor in thegrowth and repair rate of the material. The exported glucose isconverted to GL, which subsequently reacts with primaryamine-functionalized acrylamide monomers, 3-aminopropyl methacrylamide(APMA), to build a polymer matrix. The chloroplast embedded-gel matrix,containing lightly cross-linked polymer networks that swell in water,continually grows, strengthens, and self-repairs using fixation ofatmospheric CO₂ as a regeneration source under ambient illumination wasdemonstrated.

Hydrogel Composite Design and Synthesis

The three major saccharides exported from chloroplasts (extracted orin-vivo) are maltose, glucose and triose phosphate. Glucose as a reagentto focus on because it is easily converted into a reactive precursor,gluoconolactone (GL) were selected. One D-glucose molecule is oxidizedto one D-gluocono-1,5-lactone molecule and one hydrogen peroxide (H₂O₂)molecule by glucose oxidase (GOx). These scheme has been used forglycopolymer synthesis previously. The product of the polymerizationreaction of GL and aminopropyl methacrylamide (APMA) appears transparentand gel-like. The characteristic IR peaks, lactone C═O of GL appears at1719.7 cm⁻¹ and acrylamide C═O of APMA appears at 1652.9 cm⁻¹ and 1616.7cm⁻¹ in the mixture of GL and APMA at 0 h (FIGS. 2A, 6A-6F, and 7A-7B).The mixture of GL and APMA in pH 7.0 phosphate buffer is put under theambient light for 5 h until hydrogel (denoted as GPMAA) forms, asindicated by the presence of a new broad peak at 1624.3 cm⁻¹ thatcorresponds to the formation of amide bond between GL and APMA as wellas the polymerization of acrylamide (FIG. 2A). The rheologicalproperties of the hydrogels show that the storage modulus G′ at highangular frequency (100 rad s⁻¹) is 3 kPa and shear modulus measured atdifferent reaction times implies that GPMAA synthesis in completedwithin 18 h (FIGS. 2B and 8). GPMAA shows a swelling property andexhibits a 115-fold increase hydrogel weight in 48 h due to waterabsorption. Using atomic force microscopy as an indenter, the Young'smodulus of chloroplasts was estimated to be 26 (±5) kPa, which is higherthan that of the swollen GPMAA hydrogel (water 50 wt %) at 8.2 (±4.1)kPa and lower than that of dry GPMAA at 348 (±125) kPa (FIGS. 10A-10C).Thus, the chloroplasts can reinforce the GPMAA hydrogel in a liquidenvironment. In addition, graphene oxide at low weight fraction (0.01 wt%) can be used to immobilize glucose oxidase (GOx) on the surface ofgraphene oxide sheets, which also serve as mechanical inclusions forstiffening. Graphene oxide-containing hydrogel is formed within 6 h,which is 3 times faster than that of hydrogel formation without grapheneoxide (FIG. 2C). After 6 h, the shear modulus of GPMAA hydrogel is 0.4(+0.05) kPa while that of graphene oxide containing GPMAA hydrogel is3.5 (+0.19) kPa, which is approximately 9 times higher. The shearmodulus of GPMAA hydrogel at 18 h (completely formed) is 3.0 (+0.087)kPa, which is still approximately 17% lower than that of graphene oxidecontaining GPMAA. A low fraction of graphene oxide was observed tostiffen hydrogel 3 times faster by accelerating crosslinking, enhancingthe mechanical strength by 17% as the composite material is intertwinedby hydrogen bonds between hydroxyl and epoxy groups of graphene oxidesheets and GPMAA chains (FIGS. 2B and 9A-9D). The hydrogel exhibitsadhesive properties, which displays characteristic fibrillar structureand shear strength profile during the separation process (FIGS. 2C and2D) and can hold the weight of 12 mL of water in a centrifugal tube(FIGS. 11A-11C).

Engineering of Extracted Spinach Chloroplasts

Since glucose export from the extracted chloroplasts is a potentialrate-limiting step in material growth, this prompted an optimizationstudy on glucose export. Chloroplasts, plant organelles contained withinthe cytoplasm of the plant cell, are the main sites of carbon fixationand photosynthesis in plants. They have been explored as candidates forsolar energy generation and efficient carbon dioxide sequestration (100μmol CO₂ mg⁻¹ Chl h⁻¹) due to their inherent ability to export storedchemical energy, abundance in nature, and scalable isolation from plantmatter. However, they have not been used as components within materials.Exported sugars ultimately participate in the sucrose synthetic pathwaythrough a series of enzymatic reactions in the cytoplasm of protoplasts.This pathway is absent in isolated chloroplast, potentially yieldingaccumulation of exported glucose and increased availability for materialsynthesis. Therefore, extracted chloroplasts can accumulate exportedsaccharides with the absence of the sucrose synthetic route.

Several biochemical and nano-biotechnological approaches were exploredto increase the export rate of glucose from isolated chloroplasts. Underdark conditions, maltose and glucose are the major sugars exported fromchloroplasts. Maltose is a disaccharide consisting of two glucosemolecules joined with a α(1→4) glycosidic bond that α-glucosidase canhydrolyze. Therefore, α-glucosidase can be used in the chloroplastincubation medium as a means of converting the maltose to glucose andboosting the glucose yield. The glucose concentration outside of theextracted chloroplast after 2 h of light and 2 h of dark period withα-glucosidase is about 3 times higher than that of control withoutα-glucosidase (FIG. 3A). This proves that the maltose is exported fromthe chloroplast and subsequently broken down by enzymatic hydrolysis toproduce additional glucose molecules for eventual polymerization.

Starch formation and sucrose synthesis are often viewed as competitiveprocesses since starch is formed in the chloroplasts by photosynthesisduring the day and exported after being broken down to synthesizesucrose at night. Indeed, glucose concentration significantly declinesunder continuous illumination for 24 h without an intermittent darkperiod (FIG. 3B). This appears consistent with the starch degradationprocess becoming latent or a glucose influx competitively operating withglucose export. Alternatively, if stored in the dark for 24 h,chloroplasts equilibrate glucose to a constant concentration (FIG. 3B),which supports the conclusion that a prior period of illumination isnecessary before a dark period for starch formation, breakdown andglucose export.

Another important variable is inorganic phosphate (Pi) concentration. Itis known to play a key role in photosynthesis and carbon metabolism. Thephotosynthesis of isolated chloroplasts soon ceases in the absence of Pibut restarts with exogenous addition of Pi to the medium. Consequently,a Pi deficiency can limit carbon export from isolated chloroplasts. Toinvestigate the importance of external Pi concentration on glucoseexport, isolated chloroplasts are incubated in buffer containing 5 mMPi, with subsequent hourly additions of Pi to the incubation medium tomaintain an external Pi concentration. However, an insignificantdifference in external glucose concentration can be seen with this highPi supply as shown in FIG. 3B. Supplying external Pi can be anineffective strategy to increase the glucose export rate in extractedchloroplasts.

The membrane mechanisms for glucose export can also equilibrate with aninflux rate, leading to limiting glucose concentrations outside thechloroplast. As another optimization variable, the pH of the chloroplastsuspension was adjusted from pH 7.6 to pH 8.0 to mimic the protongradient between the chloroplast stroma and the external environment inthe dark. FIG. 3C shows that the change in glucose concentration isnegligible and within the error range even with the introduction ofexternal adenosine triphosphate (ATP) to induce the active transport ofglucose in the presence of 5 mM Pi. To minimize glucose influx andincrease glucose export, the external glucose concentration wascontinually lowered by converting glucose to glucose-6-phosphate byhexokinase. This results in an increase of glucose export at a rate ofapproximately 5 μg mg⁻¹ Chl h⁻¹ in the dark period (FIG. 3D). In abiological system, negative feedback is a well-known regulatorymechanism in which the formation of a product in turn reduces thedriving force for its own production. However, this is the firstdemonstration of boosting the net glucose export from isolatedchloroplasts by adjusting the glucose gradient across the chloroplastmembrane. As expected, additional Pi results in insignificantenhancement of glucose export from isolated chloroplasts.

Stabilization Through Plant Nanobionics.

Chloroplasts outside of the plant cells have limited photoactivelifetimes of less than a day and only a few hours for saccharide export.Several strategies have been attempted to extend chloroplastphotostability, for example by encapsulating chloroplast in biologicallyinert matrices and altering conditions such as illumination,temperature, and buffer composition. Previously, our group showed thatpotent antioxidant cerium oxide nanoparticles, nanoceria, could extendthe photoactive lifetime of isolated chloroplasts by scavenging reactiveoxygen species (ROS) produced as a by-product of photosynthesis.However, the effect of ROS scavenging on glucose export remainedunknown. The protection of the carbon export system from ROS-relateddegradation or photodamage can help maintain high carbon export ratesfor a longer period of time. Isolated chloroplasts are firstpre-incubated with nanoceria to allow the nanoparticles to enter thechloroplasts. After the incubation, the buffer was replaced with a freshbuffer without nanoceria and the chloroplast suspension is illuminatedfor 4 h and subsequently kept in the dark for 4 h. To boost glucoseexport, hexokinase was added every hour during the dark period. Aninsignificant effect on glucose export is observed at both low (5 μM,0.56 mg L⁻¹) and high (50 μM, ˜5.6 mg L⁻¹) concentration of nanoceria(FIG. 11C). When the light period was extended from 4 h to 12 h, 50 μMnanoceria gives a positive effect on glucose export after 6 h from thestart of dark period, while a marginal improvement is observed at 5 μM(FIG. 3E). Removing photo-generated ROS inside chloroplasts can extendthe lifetime of isolated chloroplasts, which ultimately translates intohigher glucose accumulation in the medium. Glucose export increases onlyafter the addition of hexokinase, which acts as a sink for this fluxoutside of the chloroplast.

In all, the yield of glucose can be increased using enzymatic hydrolysisof maltose exported from isolated chloroplasts. A net increase inglucose export was observed by adjusting the glucose gradient across thechloroplast membrane, optimizing the illumination period and enhancingthe photo-stability of chloroplasts. These findings can be applied toboost glucose export from isolated chloroplasts with the ultimate goalof conversion to the monomer, GL. Similar to the regulation of glucoseequilibrium across the chloroplast membrane by hexokinase (FIG. 3D), theconversion reaction of external glucose to GL by GOx promotes glucoseexport by suppressing glucose accumulation in the medium (FIG. 3F). Inthe presence of 20 U mL−1 GOx, the GL concentration notably increases ata rate of 12 μmg⁻¹ Chl h⁻¹ whereas the lower (10 U mL⁻¹) concentrationof GOx leads to negligible improvement in the glucose export rate (FIG.3F). This increase reaches to a plateau within a few hours and the GLconcentration barely increases in higher GOx (50-100 U mL⁻¹) (FIG. 11C).The fast accumulation of H₂O₂ from GOx enzymatic catalysis maycontribute to this saturation. In the presence of high H₂O₂concentration, CO₂ fixation ceases or its rate significantly diminishessince H₂O₂ and CO₂ both compete for photoreductants generated in thethylakoids of chloroplasts. From the continuous increase of GL duringthe illuminated period, the glucose gradient adjustment across themembrane can be the more critical variable that affects glucose exportthan manipulating the light/dark cycle (FIG. 11C). This brings hugebenefits to the chloroplast-embedded hydrogel growth sincephotosynthesis, glucose export and conversion, and polymerization canoccur all together under the illumination.

Carbon Fixation in the Hydrogel System

Putting these components together, a material that autonomously grows,strengthens and repairs itself in response to certain types of damagewas constructed. Isolated chloroplasts were pre-incubate with 50 μMnanoceria for 3 h at 4° C. to prolong their lifetime, and then replacethe nanoceria solution with buffer containing both APMA and GOx. It wascritical to purge the remaining nanoceria from the medium becausephotogenerated free radicals are mechanistically essential to thepolymerization and crosslinking process. Isolated chloroplasts functionwell in media containing up to 0.1% w/v APMA but show significantlylower glucose export rates at 0.4% w/v APMA (FIG. 14). GOx has beendemonstrated to maintain its catalytic activity over that period. Within6 h from the start of the illumination period, hydrogel-like materialcan be observed around the chloroplast membrane. After 18 h, thehydrogel has clearly extended to a thickness of more than 20 μm and thechloroplasts become gradually embedded in the hydrogel (FIGS. 4A and15). When the mean concentration of glucose in the medium is 5 μM, theestimated glucose concentration near the chloroplast outer membrane isapproximately 125 mM within a 100 nm distance. Therefore, theexperimental results show that hydrogel forms mostly around thechloroplast membranes rather than in non-specific locations in themedium. When GOx was immobilized on the surface of graphene oxide film,glucose conversion occurs mostly on the sites where GOx is immobilized,and the resulting GL reacts with APMA subsequently to form GPMAA on thefilm (FIG. 4B). This is consistent with a shift in reaction kineticsfrom being glucose export-limited in the former case to GOxreaction-limited at the graphene oxide in the latter case. This is not atypical composite to have reinforcement effect; rather, designed forconvenient characterization of the hydrogel distinguished from grapheneoxide or GOx. The graphene oxide can accelerate hydrogel formation bymixing isolated chloroplasts with the GOx anchored graphene oxidesuspension, as shown in FIG. 2B.

The characteristic IR peak from the newly formed amide bond in GPMAAhydrogel appears at 1625 cm⁻¹ on graphene oxide film (FIGS. 2A and 15).The increased Raman band at 1245 cm⁻¹ is tentatively assigned to v(C-N),δ (NH) in vibrational mode (amide III) (FIG. 4C). Mapping of thecharacteristic Raman bands of GPMAA based on the ratio between two bandsat 1245 cm⁻¹ and at 1290 cm⁻¹ indicates that GPMAA hydrogel formsalongside chloroplast membranes. (FIGS. 4D, 16A-16B, and 17). Thechloroplasts-embedded GPMAA hydrogel grows slowly, at a rate of 60 μm³per chloroplast in very mild conditions and can provide suitable mediato maintain the viability of the isolated chloroplasts by entrapment.The growing hydrogel can protect chloroplast membrane as a physicalsupport and scavenges H₂O₂ generated from glucose oxidation since H₂O₂can aid polymerization and crosslinking. The stability of the embeddedchloroplast activity can be a critical factor to maintain the growthrate of this material. In this study, the embedded spinach chloroplastscan be still active for more than 80 h based on our previous study. Theinherent lifetime of isolated chloroplasts is species-dependent, rangingfrom hours to months. In addition, if α-glucosidase and glucosedehydrogenase pyrrolo-quinoline quinone could be incorporated in thesystem to hydrolyze and oxidize maltose, the growing rate of thematerial would be enhanced due to higher monomer availability.

Characterization of Self-Healing Hydrogel Composite

Physically separated hydrogels are able to seamlessly recombine uponlight exposure (FIGS. 5A-5D). In the absence of additional GL, thehydrogel shows some repair of fissures but with some defects apparentupon mechanical deformation (FIG. 5A), suggesting that the materialrepair initiates from the external surface. When 5 μL of 1 M GL solutionis added to the interface of two physically separated hydrogels, thisresults in the formation of more extensively repaired gels, which canthen sustain more stringent deformation (FIG. 5B). As a negativecontrol, the separated hydrogels placed together physically but kept inthe dark overnight can be easily separated by deformation (FIG. 17).GPMAA hydrogel forms multiple hydrogen bonds with surrounding watermolecules as well as inter-hydrogen bonds between the polymer chains.Hydrogen bonds are one of the common mechanisms used in self-repairingmaterials. Since UV-irradiation energy is directly absorbed by theglucose moiety, the formation of free radicals can be involved inacrylamide polymerization or hydrogen bonding with hydroxyl groups.Physically contacting two GPMAA hydrogels on the face restores themechanical strength (shear stress) by nearly 50%, whereas the additionof GL and light exposure allow the restoration of the originalmechanical strength (FIG. 5C). This demonstration is a mimic ofchloroplast embedded-gel matrix that primarily polymerizes andcrosslinks by light and continuously grows and self-repairs by thesupply of glucose from the atmospheric CO₂ fixation (FIG. 5D). Thisself-healing mechanism is new and different to conventional self-healingrelying on non-covalent interactions or specific chemical bonds thatform or break reversibly. Glucose molecules supplied by chloroplastsrepair the local damage by exceeding its own local material balancethrough the atmospheric CO₂ fixation.

In summary, a new class of carbon fixating materials grows, strengthensand self-repairs using ambient solar fluence and atmospheric carbondioxide. This work highlights how the photosynthetic hydrogel compositesystems can be optimized, including productivity and stability ofextracted chloroplasts by controlling the illumination period,delivering antioxidant nanoceria inside of chloroplasts, and increasingchloroplast glucose export rate. Substantial improvement in themechanical property can be needed for the practical use of thisself-healing material. Further optimization of the carbon-fixatingsystem and extending the lifetime of the embedded chloroplasts willinvariably improve the growing rate and the repair efficiency of thematerial. This class of new materials will find broad utility in fieldsranging from biomedicine, material construction, or defense relatedapplications.

EXAMPLES

Isolation of Chloroplasts:

Chloroplasts isolation was performed as previously reported with aslight modification. Commercially available fresh baby spinach leaves(Spinacia oleraceae L.) were thoroughly washed with de-ionized water andthe excess water was removed. After removing the middle veins, theleaves were chopped into small pieces (approximately 0.5 cm×0.5 cm) andhomogenated by blending in HEPES buffer (30 mM, pH 7.6) containingpolyethylene glycol (Mw. 8,000, 10% (w/v)), K₃PO₄ (0.5 mM), and MgCl₂(2.5 mM) in an iced bath. The resulting homogenate was filtered throughfour layers of cheesecloth and the chloroplasts pellet was collected bycentrifugation at 4,000 rpm for 15 min at 4° C. The chloroplasts werere-suspended in the aforementioned buffer and added on top of a 40%Percoll/buffer layer to separate the intact chloroplasts from the brokenones. The intact chloroplasts were sedimented as a pellet whereas thebroken chloroplasts form a band in the Percoll layer by centrifugationat 1,700×g for 7 min at 4° C. The upper phases were carefully removed tocollect the pellet with intact chloroplasts. This chloroplast pellet waswashed with buffer to remove Percoll, and then re-suspended in buffer.

Estimation of Chlorophyll Concentration:

The yield of isolated chloroplasts is estimated by a unit chlorophyllbasis (mg of chlorophyll). The chloroplast suspension is diluted by 100times in 80% acetone and mixed well to dissolve the chloroplastmembrane. This suspension is centrifuged for 2 minutes at 3,000×g andthe supernatant is retained. The absorbance of the supernatant isdetermined at 652 nm using a Shimadzu UV-3101PC, and then multiplied bythe dilution factor (100) followed by dividing by the extinctioncoefficient of 36 to get the mg of chlorophyll per mL of the chloroplastsuspension.

$\frac{{mg}\mspace{14mu}{chlorophyll}}{mL} = {\frac{A_{652}}{36} \times 100}$

Typical chlorophyll concentrations in this study were 0.90-1.14 mg mL⁻¹.

Isolated Chloroplasts Glucose Export:

Chloroplasts suspension (10 mL) was placed in a 6 cm-diameter glasspetri dish closed with a loose glass lid to allow chloroplasts tocapture light and atmospheric carbon dioxide. Chloroplasts wereilluminated with a light intensity of approximately 200 μmol m⁻² s⁻¹photosynthetic active radiation (40 W m⁻²) using a light-emitting diodeflood lamp FL-70W (LED wholesalers).

Glucose concentration is measured by hexokinase since glucose isphosphorylated by adenosine triphosphate (ATP) in the reaction catalyzedby hexokinase. Phosphorylated glucose, glucose-6-phosphate, is thenoxidized to 6-phosphogluconate in the presence of oxidized nicotinamideadenine dinucleotide (NAD) in a reaction catalyzed byglucose-6-phosphate dehydrogenase. During the oxidation, an equimolaramount of NAD is reduced to NADH and consequently the absorbance at 340nm increases, which is directly proportional to glucose concentration.Glucose (HK) assay reagent (Sigma) is prepared, which contains NAD (1.5mM), ATP (1.0 mM), hexokinase (1.0 U mL⁻¹), and glucose-6-phosphatedehydrogenase (1.0 U mL⁻¹) with preservatives such as sodium benzoateand potassium sorbate. One U is defined as the amount that catalyzes theconversion of 1 micromole of substrate per minute under standardconditions. Glucose solutions in different concentrations are preparedto obtain a standard curve based on the absorbance at 340 nm. Reactionis carried out for 15 minutes at room temperature. The blank accountsfor the contribution to the absorbance of the sample and the assayreagents.

${{\mu\; g\mspace{14mu}{glucose}\mspace{14mu}{mL}^{- 1}} = \frac{\Delta\;{A \cdot {TV} \cdot {Mw} \cdot F}}{ɛ \cdot d \cdot {SV}}},$where ΔA is difference in absorbance between the sample and the blank,TV is total assay volume (mL), SV is sample volume (mL), Mw is amolecular weight of glucose 180.2 g mol⁻¹, F is dilution factor, ε isextinction coefficient for NADH at 340 nm (mL μM⁻¹ cm⁻¹), and d is lightpath 1 (cm).

The initial glucose concentration from isolated chloroplasts within onehour is determined to be on average approximately 130 μg mg⁻¹. Thisvalue is attributed to previously stored starch inside the chloroplastsand is therefore subtracted to exclusively study glucose export fromphotosynthesis in isolated chloroplasts. Accordingly, concentration isshown in negative value when glucose influx is higher than glucoseexport. Although chlorophyll concentration of chloroplast suspension ismaintained at approximately 0.1 mg mL⁻¹ throughout all experiments,control experiment were performed each time to account forbatch-to-batch variability in functioning chloroplasts from eachextraction round. The amount of sugar molecules exported from isolatedchloroplast has been reported as an accumulated quantity within thefirst few hours from extraction. Glucose concentration measured for 8 hbecause physical damage in the chloroplast membrane starts beingobserved after 8 h of incubation at room temperature.

Measurement of Gluconolactone:

Gluconolactone centration measurement was performed by assay kit(Megazyme Inc., Ireland) and followed the procedure. Gluconolacone (GL)is hydrolyzed in sodium hydroxide solution (2 M, pH 11) at roomtemperature for 10 min. The resulting gluconic acid is phosphorylated togluconate-6-phosphate by gluconate kinase and ATP. Gluconate-6-phosphateis converted to ribulose-5-phosphate by 6-phosphogluconate dehydrogenase(6-PDGH) in the presence of nicotinamide-adenine dinucleotide phosphate(NADP⁺). The absorbance at 340 nm was measured, which is increased bythe amount of reduced nicotinamide-adenine dinucleotide phosphate(NADPH) formed in this reaction that is stoichiometric with the amountof gluconic acid. Reaction is carried out for 6 minutes at roomtemperature. The blank takes into account the contribution to theabsorbance of the sample and the assay reagents.

${{\mu\; g\mspace{14mu}{GL}\mspace{14mu}{mL}^{- 1}} = \frac{\Delta\;{A \cdot {TV} \cdot {Mw} \cdot F}}{ɛ \cdot d \cdot {SV}}},$

where ΔA is difference in absorbance between the sample and the blank,TV is total assay volume (mL), SV is sample volume (mL), Mw is amolecular weight of gluconolacone 178.1 g/moL, F is dilution factor, εis extinction coefficient 6300 for NADPH at 340 nm (L mol⁻¹ cm⁻¹), and dis light path 1 (cm).

Nanoceria Synthesis:

Poly (acrylic acid)-coated nanoceria was synthesized by Asati et al.with some modifications. Cerium (III) nitrate (1 M, 2.5 mL, SigmaAldrich) and an aqueous solution of poly(acrylic acid) (Mw 1,800, 0.5 M,2.5 mL, Sigma Aldrich) were added dropwise to HEPBS buffer (0.4 M, 12.5mL, Sigma Aldrich). The resulting mixture was adjusted to pH 8.5 withNaOH (8 M) and the reaction was continued for 1 day at room temperatureunder magnetic stirring. The supernatant was collected, concentrated andpurified by centrifugation at 4,000 RCF for 10 min using a 10K Amiconcentrifugal filter (Millipore Inc.).

Hydrogel Synthesis:

Gluconolactone (GL) solution was mixed with 3-aminopropyl methacrylamide(APMA) solution in phosphate buffer (pH 7.0) or chloroplast buffer (pH7.6), and the mixture was placed under the ambient light for overnightat room temperature. GL (1 M) and APMA (1 M) solution were used for invitro synthesis for characterization. This mixture (70 μL) of GL andAPMA was placed on the glass slide and kept under the light in the airafter 40 min UV-irradiation at 365 nm (4 W (J s⁻¹), 5.5 cm distance).(UVGL-15, Ultra-Violet Products Let. CA, USA)

Measurement of Hydrogel Swelling Property:

Dry hydrogel (80-120 mg) was immersed in 50 mL of deionized water for 48h at room temperature. After swelling, the hydrogel was sediment toseparate the insoluble part. The selling was calculated as follows

${Swelling} = \frac{W_{s} - W_{d}}{W_{d}}$

where, W_(s) is the weight of hydrogel in swollen state and W_(d) is theweight of hydrogel in dry state.

Evaluation of Rheological Properties:

Rheological properties of the hydrogels were characterized using AntonPaar MCR-301 rheometer (Anton Paar, Ashlanad, Va., USA) operating underdisposable parallel plate geometry (10 nm diameter) at room temperature.Dynamic strain sweep (0.1-100% strain at constant 10 rad s⁻¹) wasconducted to verify the linear viscoelastic regime, and then carry outfrequency sweeps between 0.1 and 100 rad s⁻¹ at constant 1% strain.Hydrogels are swollen in 100 wt % DI water for 30 min, then loaded ontoa sand paper (Grit:P80, Norton Abrasives, Worcester, Mass., USA) toavoid slipping. All measurements were run in triplicate and the resultsare expressed as the average with standard errors.

Evaluation of Mechanical Property:

The mechanical property of the hydrogel was evaluated using 8848MicroTester (Instron Corp. Mass., USA), where two hydrogels physicallycontacted to each other was pulled apart by shear stress at the rate of0.04 mm s⁻¹. Two hydrogels were separately formed on each glass slide(15×15×0.5 mm). As partially formed hydrogels were physically contactedon the face, the hydrogel continued to polymerize and crosslink,resulting in one merged hydrogel between glass slides. The hydrogel wasclamped via sticky tape that tightly glued to the glass slide. Therepairing test was carried out in a similar way. Two complete hydrogelswere physically contacted on the face, and then mechanical force wasapplied or they were kept under the illumination after adding GL to thehydrogel interface.

Estimation of Glucose Concentration Near Chloroplast Membrane.

Approximately one chloroplast per 100 μm³ (V₁) is observed in themicroscope images with 0.1 mg Chl mL⁻¹ chloroplast suspension. When themean concentration of glucose measured in the medium is 5 μM h⁻¹,assuming a chloroplasts as a spherical organelle, the concentration ofthe exported glucose molecules within a 100 nm distance (x) in a 0.01 ms(t) period (t≈x²/2D), where glucose diffusion coefficient (D) is 5×10⁻⁶cm² s⁻¹ in water at 25° C.; V₂=4/3π(0.1)³=4×10⁻³ m³) is 2.5×10⁴ times asconcentrated as the mean glucose concentration in the medium, and istherefore estimated to be 125 mM h⁻¹.

Preparation of Hydrogel Using Chloroplasts on the Graphene Oxide Film:

GOx (20 U mL⁻¹) was mixed with graphene oxide solution (0.1 mg mL⁻¹,Graphene Supermarket, NY) for 1 h at room temperature. This mixture isdeposited on the amine-functionalized glass slides for 2 h at roomtemperature, followed by gentle washing with PBS (×3. Freshly isolatedchloroplasts are pre-incubated with nanoceria for 3 h at 4° C., and theremained nanoceria was removed by centrifugation at 4000 rpm for 5 min.The resulting chloroplasts were re-suspended (0.1 mg mL⁻¹) in 0.1% APMAcontaining buffer, and then added on GOx immobilized graphene oxidefilm, and incubated under the ambient light for 18 h.

Characterization of Hydrogel (FT-IR Spectroscopy):

Characteristic peaks of functional groups were confirmed by Fouriertransform infrared (FT-IR) spectroscopy (Thermo Electron Co. WI, USA).

Characterization of Hydrogel within Chloroplasts Suspension (FT-IRSpectroscopy):

Characteristic peaks of functional groups were confirmed by FT-IRspectroscopy (FTIR6700 Thermo Fisher Continuum FT-IR microscope). FT-IRspectra were collected from spot size 100×100 μm.

Characterization of Hydrogel within Chloroplasts Suspension (RamanSpectroscopy mapping):

Raman spectroscopy maps were collected in a confocal Raman spectrometerHR-800 (Horiba BY) using a 632 nm laser source with a 100× objective.

A Hertzian model can describe the measured force curve:

${F = {\left( {\frac{3}{4}E_{eff}\sqrt{R}} \right)h^{3/2}}},$

where F is the applied force, E_(eff) is the effective Young's modulusthat can be obtained with the following relation:

${\frac{1}{E_{eff}} = {\frac{1 - \vartheta^{2}}{E} + \frac{1 - \vartheta_{i}^{2}}{E_{i}}}},$where ϑ is the Poisson ratio (assumed to be 0.5 for the gel). Subindex icorresponds to the mechanical properties of the AFM probe (SiO₂ E!=76GPa, ϑ_(!)=0.17). R corresponds to the tip radius: the sharp probes weremodelled as cones with 7 nm base radius with 7° half-angle (Olympus,AC240TS), and the colloidal probes as 10 m spheres (NovascanTechnologies, Inc., PT.PS.SN. 10). The analysis was performed in AsylumResearch software with prior inverse optical lever sensitivity and tip'sspring constant calibrations.

Glucose concentration exported from extracted spinach chloroplasts wasmeasured using the microfluidic chip to verify (crosscheck) the system.Isolated chloroplasts (130 μg/ml) were placed in a microfluidic chamberwith a microsieve and left in the dark. Every hour chloroplasts werewashed with equal amounts of fresh buffer, while the produced glucosewas carefully collected at the outlet. Extracted glucose was measuredusing a pre-calibrated cytochrome c (cyt c)/GOx spots. Experiments wereperformed in triplicates. Chlorophyll content was found to be 5 μg/ml inextracted solution, proving successful chloroplast retention inside themicrofluidic chip.

Microfluidic Fabrication:

The 2-layered microfluidic chip was fabricated in PDMS.^([69]) Briefly,the bottom layer (100 m thickness) was formed by spin-coating PDMS at500 rpm for 40 s. This layer contained microfluidic channels, a 5×10 mm²chamber for chloroplasts, and 5 μm microsieves to prevent chloroplastsfrom moving out during washing. The top layer (5 mm thickness) hadsimilar structure except microsieves.

Glucose Sensor Fabrication:

Cyt c/GOx sensing spots were fabricated according to the previouslydeveloped procedure. Briefly, aqueous cyt c (4 mM) and GOx (500 U/ml)droplets were printed with a microarray printer using a 5 nLdelivery-volume onto porous membranes (GSWP 220 nm, Millipore). Theprinted spots were cross-linked in vaporous glutaraldehyde for 1 h under100% relative humidity and subsequently stored in water at 4° C. Glucosedetection method relies on absorption changes in cyt c spectrum. To thisend, absorption spectra were recorded in transmission mode underwhite-light illumination using 20× objective and a grating spectrometer(DU401A-BR-DD, Andor).

System II: Self Assembled Semiconducting Photocatalysts (Replacing theChloroplasts) for the Direct CO₂ Reduction to Formaldehyde and then toStable Polyoxymethylene

The chemical mechanism of System II is demonstrated in FIG. 18. Thefirst step involves the photoreduction of CO₂ to formaldehyde, which isa heterogeneous process that takes place in the solid-liquid orsolid-gas interface. The atmospheric CO₂ (gas phase or dissolved in asolution) is converted to various products in presence of aphotocatalyst (solid phase). The photocatalyst provides catalytic activesites for the reactants, absorbs light and generates electron-holepairs, transport the charges to the surface, and finally transfers theelectrons to the CO₂ for reduction reactions. Reduction of CO₂ tovarious products requires multiple electron transfers (listed below) toproduce a wide range of products. The first electron transfer reactionto CO₂ presents the highest barrier to the process. The adsorption ofCO₂ on the photocatalyst surface reduces this energy barrier andactivates CO₂ for reduction by disrupting its linear symmetry in theadsorbed state. Simultaneously, adsorption of an electron-donor (usuallywater) on the photocatalyst surface consumes the holes and prevents therecombination of electron-hole; thus, providing a constant supply ofelectrons for the reduction reactions. The number of transferredelectrons and the reaction pathways determine the CO₂ reductionproducts. The reduction mechanism is rather complicated and not fullyunderstood to this date. Experimental data suggest that at least twobranching pathways exist: (1) the formaldehyde pathway that producesformic acid, formaldehyde, methanol and methane, and (2) the carbenepathway that produces carbon monoxide, methanol, methane, and possiblyethane. In order to produce POM from CO₂, the formaldehyde productionwas maximized through the formaldehyde pathway. Minimization of thethermodynamic and kinetic barriers of the intermediate reactions anddirecting the reduction pathway toward production of more formaldehydewill increase both efficiency and selectivity of formaldehyde comparedto other products. In the second process, formaldehyde reacts in anacidic medium to form trioxane. This process is affected by the acidityof the reaction media and the temperature. In the third process,trioxane polymerization occurs in presence of an acidic initiator. Afterspecific amount of time, the polymer chains propagate and theirconcentration increases over time, yielding an increasing mass of solidproduct.

In this alternative approach, three chemical processes are combined toconvert CO₂ to polymeric product (i.e, POM): (i) CO₂ photoreduction toformaldehyde, (ii) Formaldehyde trimerization to form 1,3,5 Trioxane,and (iii) Trioxane polymerization to polyoxymethylene (POM). To increasethe efficiency of POM production, an in-depth understanding of thechemical mechanism leading to formation of this polymer is required. Akinetic model that counts for all the reactions and phenomena leading toformation of POM shed light on the kinetic barriers of POM productionfrom CO₂. FIG. 19 represents the kinetic model for CO₂ catalyticphotoreduction.

Compartment 1: CO₂ Photocatalytic Reduction to Formaldehyde

To achieve this overall model, a kinetic model for process (i) wasdeveloped. The CO₂ catalytic photoreduction is a complicated processthat involves many steps including the adsorption/desorption of CO₂ ontothe photocatalyst, electron transfer from photocatalyst to CO₂, a seriesof surface reductive reactions yielding formaldehyde as one of theproducts, and desorption/adsorption of the products. In the existingliterature of artificial photosynthesis, usually one of these steps isconsidered to be the rate-limiting step and the kinetic models aredeveloped based on only one of these phenomena. The kinetic model takesall these phenomena into account (FIG. 20).

CO₂ is a stable and chemically inert molecule. Reduction of CO₂ has ahigh-energy barrier and can only be performed in presence of a catalyst.Particularly, semiconductor photocatalysts can provide the energyrequired for the reduction of CO₂ by absorption of light andtransferring the energy to the CO₂ molecules adsorbed on their surface.Formic acid, formaldehyde, methanol, methane, ethane are some of theproducts of the carbon dioxide photocatalytic reduction. The exactmechanism of CO₂ reduction on a photocatalyst surface is still unknown.The reaction pathways, the product selectivity, and yield of eachreaction depends on many factors, including the choice of photocatalystand its bandgap, reaction setup, temperature, pH, etc. So far, thestudies of the reaction mechanism have suggested that the reduction ofCO₂ occurs through a series of single-electron transfer reactions to theCO₂. Each of these reactions involves reduction of CO₂ by transferringone electron from the surface of catalysts and H+ from the surroundingmedia to yield radical intermediates or the main products.

The production of formaldehyde has been mainly reported in presence ofTiO₂ as the photocatalysts. Moreover, it has been shown that thereduction of CO₂ in aqueous dispersions of TiO₂ lowers the energybarrier of the first electron-transfer reaction step and CO₂ activationand thus, increases the product yield. In most of the reaction set ups abatch reactor with a light source and gas inlet and outlet that was usedto perform the reaction. CO₂ is purged into the reactor until thesolution is saturated with the reactant. The solubility of CO₂ in theliquid phase depend on the CO₂ partial pressure according to the Henry'slaw; in most studies atmospheric pressure of CO₂ is used to evaluate thereduction reactions. Constant stirring of the sample facilitates theadsorption of CO₂ on the catalyst particles and prevent the masstransport limitation in this heterogeneous catalytic system. In such asystem, water is the main source of the H+. Water splitting reactionoccurs simultaneously in presence of the photocatalysts and yields H+.The pH of the system plays an important role in determining the reactionpathways, the CO₂ can be present in various carbonate forms in thesolution depending on the pH and thus, the adsorption and activationenergies required to reduce these forms of dissolved CO₂ are differentfrom each other.

Formic acid, formaldehyde, methanol, and methane are the main productsof CO₂ reduction in a reaction setup described above. Exact reactionmechanism is still a matter of controversy in the current literature andminimal kinetic data reporting the production of all these products areavailable. Various complicated reaction networks have been suggested,some of them justifying the presence of trace amount of products in thereactor. Recent DFT studied assist with narrowing down the reactionnetwork and investigating the most possible pathway with lowest energybarriers. The following reaction network may be best descriptive of thethermodynamically plausible pathway and is consistent with the productmeasurements:CO₂+2H⁺+2e→4HCOOHCO₂+2H⁺2e→CO+H₂OHCOOH+2H⁺+2e→HCOH+H₂OCO+2H⁺+2e→HCOHHCOH+2H⁺+2e→CH₃OHCH₃OH+2H⁺+2e→CH₄+H₂O

According to this reaction network, aqueous CO₂ reaction with hydrogenradicals to produce formic acid, carbon monoxide, formaldehyde,methanol, and methane. Radical intermediates that are generated insingle-electron transfer steps are steady-state species that are notstable and thus, cannot be measured with precision in the solution. Fora comprehensive kinetic modeling of the above reaction network in aheterogeneous catalytic system, the adsorption of the reactant on thecatalysts surface and desorption of products from the catalyst surfacemust also be taken into consideration and the surface reactions must bemodeled using the surface concentration of species according to the LHHWkinetic modeling approach. However, due to the lack of kinetic data inthe current literature and in attempt to avoid over-parameterizing themodel, the power-law modeling approach is used to explain the reactionnetwork using a series of individual first-order reaction:

$\frac{d\left\lbrack {CO}_{2} \right\rbrack}{dt} = {{{- k}\;{1\left\lbrack {CO}_{2} \right\rbrack}} - {k\;\left\lbrack {CO}_{2} \right\rbrack}}$$\frac{d\lbrack{HCOOH}\rbrack}{dt} = {{k\;{1\left\lbrack {CO}_{2} \right\rbrack}} - {k\;{3\lbrack{HCOOH}\rbrack}}}$$\frac{d\lbrack{CO}\rbrack}{dt} = {{k\;{2\left\lbrack {CO}_{2} \right\rbrack}} - {k\;{4\lbrack{CO}\rbrack}}}$$\frac{d\lbrack{HCOH}\rbrack}{dt} = {{k\;{3\lbrack{HCOOH}\rbrack}} + {k\;{4\lbrack{CO}\rbrack}} - {k\;{5\lbrack{HCOH}\rbrack}}}$$\frac{d\left\lbrack {{CH}_{3}{OH}} \right\rbrack}{dt} = {{k\;{5\lbrack{HCOH}\rbrack}} - {k\;{6\left\lbrack {{CH}_{3}{OH}} \right\rbrack}}}$$\frac{d\left\lbrack {CH}_{4} \right\rbrack}{dt} = {{k\;{6\left\lbrack {{CH}_{3}{OH}} \right\rbrack}} - {k\;{7\left\lbrack {CH}_{4} \right\rbrack}}}$

where t is the reaction time and k1 to k6 are the reaction rateconstants for the above-mentioned reactions. The reaction rates can befound by fitting the model to the experimental kinetic data from Penget. al. that reported the product yield for formic acid, formaldehyde,methanol and methane over time. As shown in FIG. 20, the model is highlyconsistent with the experimental data with minimal error. To assure theprecision of the model, the confidence intervals of the fitted rateconstants were calculated by perturbing the experimental data input by5% (Table 1). The calculated rate constants are shown in the belowtable. The first reaction of CO₂ reduction to carbon monoxide and formicacid have considerably smaller rate constants due to the higheractivation energy of the CO₂ compared to other product in the reactionchain.

Compartment 2: Formaldehyde Conversion to 1,3,5-trioxane

Production of trioxane from formaldehyde has been studied in theliterature to some extent. The reaction can proceed in concentratedaqueous solution of formaldehyde and in presence of a Lewis acid as thecatalyst. The reaction is known to be slow at room temperature andinvolves many unstable intermediates. Thus, in industrial plants thereaction is usually carried at temperatures above 100° C. The kineticdata available in the literature are usually collected at such hightemperatures and thus, the rate constants for the reaction at the roomtemperature is unknown. Although the reaction involves manyintermediates, the overall conversion of formaldehyde to trioxane can bedescribed by the below reaction:

To estimate the rate constant of this reaction at room temperature apower-law model can be used to describe the rate of the overallreaction:

${\lbrack\mspace{14mu}\rbrack\frac{{d\left\lbrack {C_{3}H_{6}\; O_{3}} \right\rbrack}\;}{dt}} = {{k\;{1\lbrack{HCOH}\rbrack}^{3}} - {k\;{2\left\lbrack {C_{3}H_{6}O_{3}} \right\rbrack}}}$

where t is time and k1 and k2 are the reaction rate constants for theforward and the reversible reaction, respectively. The kinetic data fromliterature at different temperatures (360, 373, 380 K) were used toestimate the activation energy of this reaction according to theArrhenius law and then calculate the reaction rate constant at roomtemperature, k1=2.31×10⁻¹² and k2=1.64×10⁻¹⁰. Using these rate constantsand assuming that all the formaldehyde produced in Compartment 1 isextracted, concentrated, and transferred to Compartment 2 uponproduction, trioxane can be produced with minimal yield as depicted inFIG. 21. Obviously, the formaldehyde conversion to trioxane is very slowat room temperature. In order to produce considerable amount of trioxanefrom photocatalytically produced formaldehyde, strategies to acceleratethe reaction using more efficient catalysts or temperature rise suingthe light source with minimal energy input must be further investigated.

Compartment 3: Trioxane Polymerization

Moreover, a kinetic model was developed for process (iii) that explainthe formation of POM from trioxane. This reaction has been studied tosome extent in the literature; it is known that two phases exist forthis polymerization reaction: (1) an induction period in which thetrioxane reacts to produce tetraoxane and minimal polymerization occurs,and (2) the secondary phase in which the POM propagates and itsproduction rate increases over time upon constant supply of thetrioxane. While various kinetic models have been proposed for theinduction period, there has not been a kinetic model reported for thesecondary phase. The model takes into account the concentration ofinitiator and the trioxane concentration in production rate of thepolymer in the secondary phase. A model was developed for process (ii)based on the experimental data for this reaction, and use these threekinetic models to develop an overall model for POM production fromatmospheric CO₂.

Modeling of Compartmental Reactions in Systems I and II for Optimizationof the Systems

The overarching goal of this study is to exploit ambient solar energyharvesting and carbon dioxide reduction to create a new class ofregenerative, densifying materials—a class that literally grow in CO₂and sunlight. This class of materials point to several fundamentalquestions relating to carbon fixation and its incorporation intofunctional materials. By performing these reactions within materialcompartments, it is possible to create coatings and supports thatcontinuously grow and self-repair using carbon dioxide as a carbonsource. Such materials would significantly benefit transportation andconstruct costs, and exhibit self-healing and densification over time.Significant progress has been made to date on two systems.

Mathematical Modeling of Spatial and Temporal Densification Matrix inSystem I

Mathematical modeling of metabolism is a powerful tool for gainingsufficient understanding of complex reaction and metabolic pathways forthe optimization of biologically-based system design. In the case ofsystem I, a metabolic model of C₃ leaf carbon metabolism is used for theproduction of carbohydrates, which then be used as source terms for aspatiotemporal transport model of polymer production within the proposedchloroplast-entrapped hydrogel matrix. This type of modeling isfundamentally necessary, as the continual densification of the matrixposes growing diffusion barriers, resulting in a tapering of overallfixation rates.

The modeling consists of two stages: (1) chloroplast carbohydrateproduction and export, (2) conversion and polymerization within theengineered hydrogel matrix. Pertaining to the former stage, whileextensive and detailed mathematical models of C₃ leaf carbon metabolismexist, an experimentally based transfer function approach was taken. Inthe case of the plasmid chlorophyll, a transfer function with the inputsbeing CO₂ concentration and light exposure was constructed, and theoutputs being the flux of maltose and glucose as a function of time(FIG. 22). As inputs and outputs can both be measured, a directanalytical solution or a neural-network based approach is employed. Thisfunction system was improved by scanning the input space experimentallyby varying light exposure and CO₂ concentrations. As using livingsystems in CO₂ fixation is complex, especially the plasmid based diurnalconversion and transport, successful completion of this model present asignificant advance in the ability to predict and study the dynamics ofutilizing chlorophylls in the manufacturing setting. This work is inoptimizing the conditions of biomass densification, and potentiallymotivates significant improvements in scaffold design.

TABLE 1 The reaction rate constants for CO₂ photocatalytic reduction inpresence of TiO₂ estimated from fitting the power-law kinetic model toexperimental data and the associated errors. Error within 95% RateConstants confidence interval of the (1/s) experimental data k1 3.31 ×10−6 1.60 × 10−16 k2 1.55 × 10−7 3.97 × 10−14 k3 2.46 × 10−5 2.18 ×10−12 k4 1.42 × 10−5 2.54 × 10−12 k5 1.67 × 10−4 1.25 × 10−10 k6 6.93 ×10−4 2.47 × 10−9 

More generally, production of high-energy materials from CO₂ withnegative carbon footprint using renewable energy sources can be vitalfor a sustainable future.

A polymeric material that can (i) grow, (ii) densify, and (iii)self-heal over time upon exposure to sunlight and atmospheric CO₂ is anovel class of biomimetic materials with unique production processcharacteristics: minimal energy cost and negative CO₂ footprint. Here, aCO₂ fixing pathway through atmospheric CO₂ conversion topolyoxymethylene (POM). POM is an engineering plastic mainly used in theautomotive and electronics industry for its unique chemical stabilityand mechanical properties was investigated. A self-healing anddensifying POM composite generated from atmospheric CO₂ will bring newopportunities for the production of protective coatings and structuralcomposites. This novel class of POM composites may be realized by anoverall pathway featuring a compartmental catalytic system consisted of:(a) photocatalyst; (b) monomer formation catalyst; and (c)polymerization initiator exposed to atmospheric CO₂ and sunlight. Here,each reaction unit to identify the rate-limiting steps was kineticallymodeled using available data in the literature. the key catalytic andphotocatalytic key reactions necessary for maximizing the POM growthrate can be identified. Further, the maximization of POM growth ratethough reaction engineering strategies and enhancing the carbonadsorption capacity was investigated. Finally, the regimes of reactionkinetics and CO₂ adsorption capacity which deliver the desired andmaximized POM growth rate can be determined.

The chloroplast in plants uses the solar energy to fix atmospheric CO₂into glucose (and other form of sugars). Further polymerization ofglucose yields biomass in form of cellulose, starch, etc. Artificialphotosynthesis, is focused on mimicking the first half of plantsfunction by reducing CO₂ to hydrocarbons and fuels, utilizing solarenergy with the aid of photocatalysts. Like in plants, the CO₂ reductionreaction is accompanied by water splitting reactions on thephotocatalyst surface to provide the electrons required for the CO₂reduction reactions. However, the low CO₂ conversion rates and pooryield and selectivity of products necessitates the experiments to becarried under higher CO₂ pressures (usually 1 atm of pure CO₂), even forthe most successful artificial systems. The conversion of actualatmospheric CO₂ with a partial pressure of 400 ppm is one element ofartificial photosynthesis missing in current research efforts. Anothermissing element in artificial photosynthesis is mimicking the secondhalf of the plants function in polymerizing the elementary CO₂ reductionproducts to higher-energy, more complex structures that can grow anddensify over time.

A polymeric macromolecule that can (i) grow, (ii) densify, and (iii)self-heal over time upon exposure to sunlight and atmospheric CO₂ is anovel class of materials that may be recognized by its main productionprocess characteristics: minimal energy cost and negative CO₂ emissionfingerprint. Using renewable energy source, abundant reactants,earth-abundant photocatalysts (such as graphitic carbon nitride) makethis product more economic and simultaneously eco-friendly. Thus, thisproduction strategy brings new opportunities in chemical productionindustry, structural composites, and protective coatings. Moreover, theminimum energy and transportation cost of this process make iteconomically more competent among the state of the art thermo- andelectro-chemical reduction processes for CO₂.

Devising a reaction pathway consisting of two catalytic compartments for(i) photocatalytic conversion of atmospheric CO₂ to elementary productsand (ii) polymerization of CO₂ reduction products is the first steptoward realizing this novel class of materials. Extracted chloroplastcan be coupled with a secondary polymerization chemistry to produceself-healing polymeric materials only using atmospheric CO₂ and light asenergy source. However, the CO₂ reduction to glucose is restricting (themechanism of CO₂ reduction by chloroplast always yields glucose as thestarting point for the final product).

Replacement of chloroplast with photocatalyst expands the number offeasible pathways toward novel polymeric product because it yieldsmultiple CO₂ reduction elementary products such as formic acid,formaldehyde, carbon monoxide, methanol, and methane. Additionally,coupling the photocatalytic compartment with the secondarypolymerization compartment can extend the life time of the catalyticsystem, avoiding concerns such as chloroplast short life span or damageupon exposure to harsh atmosphere. However, the low conversion rate ofCO₂ in photocatalytic systems (˜few micromole/hr of products) is achallenge that will affect the yield and growth rate of final polymericproduct. Hence, any proposed pathway from atmospheric CO₂ to a polymericproduct must be evaluated for their thermodynamic feasibility andkinetics of reaction.

Here, a thermodynamically feasible pathway from CO₂ to polyoxymethylene(POM) using kinetic engineering was evaluated. This pathway consists oftwo main reactions: (i) photocatalytic reduction of atmospheric CO₂ toformaldehyde and (ii) catalytic polymerization of formaldehyde to POM.POM contains repeating units of oxymethylene (—O—CH₂—) and is producedindustrially by polymerization of 1,3,5-trioxane (C₃H₆O₃), a stablecyclic trimer of formaldehyde. Therefore, a full chemical pathwayconsists of three main compartments (FIG. 23): (1) CO₂ reduction toformaldehyde, (2) formaldehyde conversion to 1,3,5-trioxane, and (3)trioxane polymerization to POM.

For each step, a reaction mechanism based on the data previouslyreported in the literature and use the kinetic data to obtain thereaction rate constants in each step was proposed. Next, the reactionsfrom each step were integrated into an overall reaction pathway from CO₂to POM to evaluate the kinetics of the process. The POM yield and growthrate are calculated and used to determine rate limiting steps of theproposed pathways. Also, the required relative improvement of thekinetics of industrial process for obtaining plausible POM growth ratesare calculated. Combining this compartmental catalytic system with CO₂capturing technology is discussed in order to provide a roadmap for theefforts focused on kinetics improvement and enhancement of CO₂adsorption.

This specific pathway is a case study to obtain further insight towardthe overall strategy of production of macromolecular products fromatmospheric CO₂. Certainly, other chemical pathways may be proposed andtheir thermodynamic and kinetic feasibility can be investigated infuture. Pathways from formic acid, formaldehyde and methanol towardethylene, acrylic acid, and simple sugars are among the feasible overallcompartmental catalytic systems that can serve as building blocks forhigh-energy polymeric materials.

Analysis and Discussion:

In the following sections, the reaction pathway proposed for eachcompartment, kinetic models fitted to the experimental data available inthe literature, and estimated/calculated reaction rate constants at25-30° C. for all three compartments is presented.

RXN1: Photocatalytic Reduction of CO₂ to Formaldehyde.

The experimental data provided by Liu et al. was used in this section(FIG. 24A). In the majority of the existing experimental literature,methanol and methane are mentioned as the main products of thisphotocatalytic process over TiO₂, the benchmark photocatalyst for thisprocess. Less kinetic data is available for formates as products and/orintermediates, mainly because of the focused interest in methanol andmethane as fuels and precursors for other chemical processes. Also, thedifficulty of distinctive measurement of formates using commontechniques such UV-vis spectroscopy is another reason to overlook thedata for these products. The dataset chosen for the calculations has theadvantage of reporting kinetic data for most prominent intermediate andproducts of the CO₂ reduction including formic acid, formaldehyde,carbon monoxide, methanol and methane. Additionally, the higher yield offormates reported in this dataset makes it of particular interest forthe proposed overall pathway toward POM.

Photocatalytic reduction of CO₂ consists of a series of deoxygenationand hydrogenation reactions occurring through multiple electron andproton transfer steps. The concurrent photocatalytic water splittingreaction provide the hydrogens required for the reduction reactions andthe photocatalyst provides the required electrons. Catalyst structureand morphology (type and availability of active sites, mode ofadsorption of reactants on the surface, size and position of thesemiconductor bandgap) and reaction condition (light source,temperature, pH, feed composition, presence of hole scavengers)determine the reaction mechanism. Reactions proceeds through different,sometimes competing, pathways and yield various products with differentselectivity including formic acid, formaldehyde, carbon monoxide,methanol and methane, etc.

Many pathways have been proposed due to the wide range of experimentallyobserved intermediates and products. Among those, formaldehyde pathwayor fast hydrogenation pathway follows CO₂→HCOOH→HCHO→CH₃OH→CH₄. Whilethis pathway is thermodynamically feasible, the kinetic models based onthis mechanism are less explored as usually they cannot explain theconcentration profiles of methanol as an intermediate toward methane.The alternative carbene pathway which proceeds as CO₂→CO→C^(•)→CH₃^(•)→CH₃OH/CH₄ better explains the concentration profiles of methanoland methane, but cannot justify the presence of observed formates in theintermediate/product spectrum. It is plausible that different reactionspathways can occur in a system, however, some of them become moredominant in specific reaction condition and in presence of specificcatalyst structures.

The experimental system under investigation by Liu et al. was saturatedwith 1 atm CO₂ in presence of 25 g of TiO₂ nanoparticles in 100 mlwater. Sodium hydroxide (0.15 was added to act as hole scavenger andpromote catalyst activity. A mercury UV lamp was used as the source oflight and the reaction was carried at room temperature. The system wasstirred during the reaction and the concentration of products weremeasured over 50 hours of reaction with 10 hours intervals. The chemicalpathway and reaction rate constants were fit to the experimental dataassuming a system limited by the surface reactions: ignoring anylimitations in mass transfer, electron transfer, andadsorption/desorption of reagents, intermediates, and products. SinceCO₂ and hydrogen evolution (water oxidation reaction) data was notreported in this study, a surface Langmuir-Hinshelwood model was notused and instead used bulk product concentration in the first order rateexpressions to avoid overfitting the data and over parameterizing thekinetic model. Also, abundant proton was assumed to be provided in thesystem through the water oxidation reaction such that its concentrationcan be considered constant. lastly, the concentration of CO₂ in theliquid phase was calculated using Henry's law.

Fitting of various kinetic models to the experimental data and parameterestimation was performed in a single step using multi-objectiveoptimization. The details of the objective function optimization areavailable in the supplementary information. Individual reaction rateswere expressed as functions of the chemical concentrations andexpressions for overtime concentration change of reactant and productswere defined. Parameters were constrained in a range of 10⁻¹⁰ to 1 (1/s)and fitting was performed by simultaneous calculation and minimizationof the following objective function using the ordinary least square(OLS) difference between the values of the experimental concentration ofeach products (C_(exp))_(i) and the modeled one (C_(model))_(i). Theoptimization algorithm was coded in MATLAB.

Among several reaction networks fitted to the data, including theformaldehyde and carbene pathways, the following reaction network(R1-R6)

with concentration expressions including first-order reactions (eq1-eq7)fitted the data best (FIG. 24A).d[CO₂]/dt=−k ₁[CO₂]−k ₂[CO₂]  (eq. 1)d[HCOOH]/dt=k ₁[CO₂]−k ₃[HCOOH]  (eq. 2)d[CO]/dt=k ₂[CO₂]−k ₄[CO]  (eq. 3)d[HCHO]/dt=k ₃[HCOOH]+k ₄[CO]−k ₅[HCHO]  (eq. 4)d[CH₃OH]/dt=k ₅[HCHO]−k ₆[CH₃OH]  (eq. 5)d[CH₄]/dt=k ₆[CH₃OH]  (eq. 6)

This pathway contains a series of irreversible reactions in which twoelectron and protons are transferred to the reactant at each step. Thetwo-electron transfer steps have been extensively studied in thetheoretical studies of CO₂ reduction and it has been shown that theyhave lower energy barriers than single-electron transfer steps. Thecompeting pathways from CO₂ to formaldehyde and CO lead to the formationof formaldehyde as an intermediate toward methanol, which issubsequently reduced to methane. Such reaction pathway has not beenproposed in the literature and definitely not fitted againstexperimental kinetic data, however, Ji et al. have predicted that apathway from CO₂ to formic acid and CO and from these product toformaldehyde has a lower energy barrier compared to well-establishedformaldehyde and carbene pathways. This new proposed mechanism canaccurately fit the high formic acid and formaldehyde concentrations inthis dataset while explaining the low methanol and methaneconcentrations. Neither of carbene and formaldehyde pathways and theircombinations with and without reversible reactions at different stepscan fit the data properly. However, one must remember that CO₂photocatalytic reduction is a complicated process that depends on manyaspects of the reaction condition and catalyst surface and thismechanism may be the dominant mechanism only in the specificexperimental conditions that this data was collected at.

The estimated rate constants and their confidence intervals are shown inTable 2. The initial reduction of CO₂ to either formic acid or carbonmonoxide is the rate-limiting step in the presented reaction pathway.The rate constants for the reductions of formic acid and CO toformaldehyde are three orders of magnitude larger. It is emphasized thatthe reduction of formaldehyde is comparably fast; as such, formaldehydeis expected to be a stable intermediate in the process, which mayexplain the lesser number of articles reporting observation of thischemical as a product.

RXN2: Formaldehyde Conversion to Trioxane.

Trioxane is industrially produced by acidic catalytic distillation ofaqueous formaldehyde solution. This well-established chemical processsuffers from high energy and low yield, with selectivity towardsbyproducts such as methyl formate and methyl glycols. In literature, thereaction is most often performed in batch at higher temperatures and thekinetic data are fitted to the overall reaction of 3HCHO→C₃H₆O₃. Inreality, the reaction network involves hydration, oligomerization, andcyclization reactions of formaldehyde and other intermediates-requiringa more comprehensive reaction mechanisms to produce an accurate kineticmodel.

In the overall pathway from CO₂ to POM, trioxane must be produced atroom temperature to avoid excessive energy input. The kinetic dataavailable for trioxane reaction at 100° C. to find the relevantparameters at room temperature was used. The experimental data oftrioxane formation reported by Yin et al. is presented in FIG. 24B. Tocapture the complicated pathway to trioxane formation and obtain moreaccurate rate constants at room temperature the following reactionpathway were used, consisting of hydration of formaldehyde (R7),dimerization (R8) and trimerization (R9) of formaldehyde, and finally acyclization reaction (R10) to produce trioxane from the linear trimer:HCHO+H₂O

HO(CH₂O)H  (R7)2HO(CH₂O)H

HO(CH₂O)₂H  (R8)HO(CH₂)H+HO(CH₂O)₂H

HO(CH₂O)₃H  (R9)HO(CH₂O)₃H

(CH₂O)₃+H₂O  (R10)

The following concentration expression based on elementary reactionswere used to describe the concentration profiles of the reactant,intermediates, and product and the water concentration was assumed to beconstant.d[F]/dt=−k ₇[F]+k _(r7)[HF]  (eq. 7)d[HF]/dt=−k ₈[HF]² +k _(r8)[D]−k ₉[HF][D]+k _(r9)[T]  (eq. 8)d[D]/dt=k ₈[HF]² −k _(r8)[D]−k ₉[HF][D]+k _(r9)[T]  (eq. 9)d[T]/dt=k ₉[HF][D]−k _(r9)[T]−k ₁₀[T]+k _(r10)[Trioxane]  (eq. 10)d[Trioxane]/dt=k ₁₀[T]−k _(r10)[Trioxane]  (eq. 11)

The values of the rate constants at 25 C were calculated/estimated andare listed in Table 2. The reversible rate constants of reaction 7-9were obtained from Ott et al. and Winkleman et al. at 360-371.15 C, theforward reaction rate constants were calculated using the equilibriumconstants reported by Kuhnert et al. at 360-371K, and the rate constantsfor the reversible cyclization reaction were fitted to the experimentaldata of trioxane by Yin et al. at the same temperature Knowing the rateconstants of the cyclization reaction at 371, the energy barrierestimated by Kua et al. using Density Functional theory calculations wasused to obtain the rate constants at the room temperature. The rateconstants for hydration and oligomerization reaction at room temperaturewere calculated using the same methodology.

The rate-limiting step in conversion of formaldehyde to trioxane is thecyclization reaction, having a rate constant that is 4-5 orders ofmagnitude smaller than the hydration and oligomerization reactions.While the polyglycol oligomers were experimentally observed at lowertemperatures, formation of the trioxane only at higher temperaturesconfirms the cyclization reaction as the main bottleneck in formation oftrioxane. At room temperature, the cyclization reaction (R10) imposes ankinetics as slow as that of CO₂ conversion to formic acid and CO and canequally affect the kinetics of the overall pathway toward POMproduction.

RXN3: Trioxane Polymerization.

POM is mainly produced through cationic polymerization of trioxane inpresence of an initiator or copolymerization with co-monomer. While bulkpolymerization occurs faster, the solution polymerization has been usedmore often to study the kinetics of process. Solution polymerizationproceeds through multiple steps of initiation, chain growth, sidepolymerizations, termination, and chain transfer. Most studied system isthe cationic polymerization in presence of acidic boron trifluoride(BF₃). In this polymerization process, the induction phase occurs fast,leaving the chain growth phase as the rate-limiting step. The inductionperiod has been extensively studied in the literature with severalreaction mechanisms proposed for this period. Conversely, few kineticdata sets and proposed reaction mechanisms exist for the rapid growthphase, as this phase is accompanied with phase separation andcrystallization of insoluble long polymer chains. Therefore, it has beenmore convenient to report the total trioxane conversion and notproduction of the final polymer and its relevant details.

The chain growth phase of the reaction into consideration for theoverall CO₂ to POM pathway was taken, as this phase dictates the rate ofproduction of the final polymer. The trioxane conversion data reportedby Shieh et al. (FIG. 24C) was used to fit the overall cationic chaingrowth reaction (R11):

with the rate expression of:d[Trioxane]/dt=−k ₁₁[I][Trioxane]²  (eq. 12)

Shieh et al. carried the experiments for this kinetic dataset at 30 Cusing BF₃ as initiator in an organic solvent. They proposed a kineticmodel that emphasizes on the crystallization and depolymerization stepsand their rate constants depended on the initial monomer concentration.On the other hand, the proposed rate expression reflects the kinetics ofchain propagation phase in a cationic polymerization process and countsfor the initiator effect and monomer concentration. Total polymerproduction, regardless of the consecutive crystallization process, isthe output of the model and moreover, the rate constant remainsindependent of the monomer concentration.

The trioxane polymerization follows a second-order reaction with respectto the monomer and a first order reaction with respect to initiatorconcentration. Commonly in the literature, the initiator initialconcentration is about 2-3 orders of magnitude lower than trioxaneconcentration to assure the formation of longer chains in thischain-growth polymerization process. While the rate constant of thisreaction at 30 C (Table 2) is generally a few orders of magnitudeslarger than the trioxane cyclization reaction (R10), this reactionproceeds slowly at lower trioxane concentrations due to the dependenceon the square of the trioxane concentration and low quantity ofinitiator. Therefore, depending on the trioxane initial concentrationeither of the trioxane formation (R10) or polymerization (R11) can bethe main rate-limiting step for conversion of formaldehyde to POM.

Overall Compartmental Reaction from CO₂ to POM.

The overall reaction pathway from CO₂ to POM includes reaction R1-R11 asshown in FIG. 25. This reaction mechanism assumes the simultaneousproduction and consumption of formaldehyde and trioxane as intermediatestoward POM in the overall pathway. The rate constants indicated in Table2 in FIG. 25 were used to evaluate the rate of production of POM undercontinuous supply of CO₂ over time. To capture the polymer productionfrom trioxane conversion, any byproduct formation is neglected, and thefinal polymer is taken to be a chain of 500 repeat units, equivalent toa molecular weight (M_(w)) of ˜45,000 g/mol (averaged over theliterature data for POM molecular weight obtained at various synthesisconditions. Any discussion or incorporation of a molecular weightdistribution of the products is beyond the scope of this work.

Such a kinetic model can be extremely limited by two main bottlenecks:first, the formaldehyde consumption in competing pathway toward methanolproduction instead of trioxane production and polymerization to POMproduction. Kinetic engineering is required to prevent the pathway fromformaldehyde to methanol and instead favor the formaldehyde conversionto trioxane. Engineering the reaction media through pH adjustment atlower acidic values (favoring the pathway toward trioxane), minimizinghole scavenger concentration such that less electron-hole pairs areavailable for the methanol pathway are examples of such kineticengineering strategies. More importantly, aqueous formaldehyde solutionsusually exist in equilibrium with methanol in bulk; thus, the additionof minimal amount of methanol at the beginning of reaction encouragesthe reversible methanol to formaldehyde reaction. The dependence of thetrioxane polymerization on the second power of the trioxaneconcentration can also slow down the polymerization process. In cationicpolymerization of trioxane such kinetic dependence is inevitable,however, other polymerization routes can be explored to overcome thisbarrier in the overall scheme of the reaction. Second major bottleneckin the overall system is imposed by the slow CO₂ photocatalyticreduction and formaldehyde formation. Engineering catalytic surfaceswith enhanced photocatalytic activity and selectivity for formaldehydepathway is another important factor in achieving the proposed overallpathway toward POM.

Maximization of POM Growth Rate Via Kinetic Engineering and CO₂Adsorption.

The effects of kinetic enhancement of each reaction unit on the overallgrowth rate of POM is shown in FIG. 26A. At atmospheric CO₂ pressure,engineering RXN2 and RXN3 units such that it overcomes the methanolproduction pathway accelerates the overall kinetic of the process untilit reaches the steady state. After this point, the overall process canonly be improved by improving the kinetics of RXN1 by engineering thephotocatalyst for enhanced CO₂ reduction activity. Therefore, RXN2 and 3mainly constrain the time to reach steady state growth rate andphotocatalytic activity determines the upper limit of the POM growthrate at any given CO₂ pressure.

So far, the POM growth rate has been calculated assuming theavailability of atmospheric pressure at 400 ppm. However, it is feasibleto increase the CO₂ concentration available at the photocatalyst surfaceby combining this compartmental catalytic unit with the CO₂ capturetechnologies. FIG. 26B indicates the POM growth rate achieved by thekinetic engineering of RXN1, 2, and 3 at higher CO₂ concentrationsenabled by various capture mechanisms after one month of reaction. TheCO2 inflection point in this graph separates two regimes: (i) belowCO_(2_inf) where the trioxane formation and polymerization (RXN2 & 3)are the main-rate limiting steps and (ii) above CO_(2_inf) where the POMgrowth rate scales linearly with photocatalytic activity and CO₂concentration. Hence, any hybrid catalytic/capture system designed forCO₂ to POM conversion can be evaluated using CO_(2_inf) as a metric; ifeffective concentration of CO₂ is below CO_(2_inf) the efforts must befocused on engineering the kinetic so the trioxane formation andpolymerization and if it is above the CO_(2_inf) enhancing the capturecapacity and photocatalytic activity is of higher priority.

To obtain a better perception of the required reaction engineeringrequired for each system, the CO_(2_inf) was mapped with respect toenhancements in polymerization (RXN3) and trioxane formation (RXN2) aswell as photocatalytic activity (RXN1) and overall formaldehydeconversion to POM (RXN2 & 3) in FIG. 27. This map can be used a sguideline such that by knowing characteristics of the reaction system(e.g., CO₂ concentration, kinetics of polymerization, photocatalyticactivity), other aspects of the system must be improved can bedetermined. Therefore, reaction engineering can be focused toward eitherdebottlenecking the kinetics of a specific compartment or improving theCO₂ adsorption by in the catalytic unit.

Strategies for Enhancing the CO₂ Reduction to Monomers Via CouplingPhotocatalyst with Solvent-Induced Electricity and/or ColloidalBatteries:

Traditional photocatalytic reduction of CO₂ with semi-conductivematerials utilizes solar energy as the sole energy source, but the slowreaction kinetics and lack of product selectivity withholds it frombeing an industrially viable solution for carbon fixation. Limitedelectron transfer from the semiconductor to the CO₂ is one of the mainreasons for slow reaction rates and low product yield. A novel hybridparticulate photo-electro-catalytic platform can combine ambient solarenergy harvesting with energy derived from a newly discoveredsolvent-nanomaterial electrical coupling or a colloidal battery that isdispersible in solvents. This platform consists of (1) a semiconductingphotocatalysts and (2) a Janus carbon particle capable of electronicgeneration through a process termed Asymmetric Chemical Doping (ACD)and/or a micron-sized.

The pure photocatalytic transformation can be augmented using thesolvent-derived electrical potential generated from ACD, establishing ahybrid process still untethered to external electrical inputs butpotentially with much higher reactivity. Asymmetric Chemical Doping(ACD) utilizes a chemical potential gradient across a single-walledcarbon nanotube network (SWNT), established via solvent molecular doping(e.g., CH₃CN or H₂O), as means of electricity generation. In thisprocess, the broken spatial symmetry in the Fermi levels of electricalcarriers inside the SWNT network translates directly into a voltagepotential. By coupling semiconductor photocatalyst with engineered SWNTparticles capable of generating electron flow through the ACD process,the hindered and low-rate electron transfer to CO₂ can be overcome. Withthe photocatalyst-SWNT interface properly tuned, this hybrid system cancreate a high-rate electron transfer pathway to CO₂ molecules, therebyimproving CO₂ reduction kinetics. Moreover, interfacing thephotocatalysts with ACD-enabled SWNT particles creates additional activecatalytic sites that allow us to alter or more precisely control thereaction pathways, and hence increase the selectivity of some productsover the others.

Similarly, micron-sized colloidal batteries can be interfaced with thephotocatalyst to facilitate the electron transfer to CO₂ and subsequentreduction reactions. The additional electrical potential prevents theelectron-hole recombination and lowers the overpotential of thereduction reactions. To power these colloidal electronic state machines,“colloidal batteries”, which are fabricated onto particles about 100 μmin size. The current version is based on metal-air battery, which iseasy to fabricate and use, and has high energy density. An active metalserves as anode, while oxygen gas is the cathodic active material. Thecolloidal batteries can be fabricated with many different methods andhave open circuit voltage around 1 V, short circuit current densityabout 0.5 mA/cm². These colloidal batteries can be dispersed in solutionor potentially fixed in a hydrogel network.

Alternative Pathways from CO₂ to Other Carbon-Fixing Polymer Composites:

As mentioned earlier, the specific pathway from CO₂ to formaldehyde andto POM is a case study to obtain further insight toward the overallstrategy of production of macromolecular products from atmospheric CO₂.Certainly, other chemical pathways may be proposed and theirthermodynamic and kinetic feasibility can be investigated. Pathways fromformic acid, formaldehyde and methanol toward ethylene, acrylic acid,and simple sugars are among the feasible overall compartmental catalyticsystems that can serve as building blocks for high-energy polymericmaterials.

Formaldehyde produced from the photocatalytic reduction of CO₂ can alsoserve as a building block of several resinous polymer materials. Underreaction with urea, formaldehyde will produce Urea formaldehyde (UF),also known as urea-methanal, a thermosetting polymer used in buildingmaterials such as particle and fiber board as well as in foaminsulation. In addition, this polymeric material serves as a nitrogensource for slow-release fertilizers.

Another class of materials enabled through this production offormaldehyde are phenol formaldehyde resins (PF) or phenolic resins.Formaldehyde, upon reaction with phenolic compounds forms a resinousmaterial. This finds use as a building material to produce laminates offiberglass and paper as well as to increase the chemical and temperatureresistance of plywood.

Additionally, formaldehyde can undergo an autocatalytic reaction knownas the formose reaction to produce C5 and C6 sugarmolecules-specifically glucose. Under basic conditions and in thepresence of a divalent cation, these sugar molecules are formed. Thispresents a chemical pathway that mirrors natural photosynthesis, bycreating formaldehyde through the photocatalytic reduction ofatmospheric CO₂, and the subsequent production of sugar molecules fromthis formaldehyde, it would be possible to produce structuralsaccharides (such as amylose) as well as foodstuff (such aspolydextrose) from ambient sources of carbon and sunlight.

The following references are incorporated by reference in theirentirety.

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Details of one or more embodiments are set forth in the accompanyingdrawings and description. Other features, objects, and advantages willbe apparent from the description, drawings, and claims. Although anumber of embodiments of the invention have been described, it will beunderstood that various modifications may be made without departing fromthe spirit and scope of the invention. It should also be understood thatthe appended drawings are not necessarily to scale, presenting asomewhat simplified representation of various features and basicprinciples of the invention.

What is claimed is:
 1. A method of self-healing a polymer matrixcomprising: exposing a polymer matrix including a catalyst to carbondioxide and an energy source; and generating additional material to thepolymer matrix from the carbon dioxide.
 2. The method of claim 1,wherein the catalyst includes a chloroplast, a nanocatalyst, or acolloidal battery.
 3. The method of claim 1, wherein the polymer matrixincludes a chloroplast in a hydrogel.
 4. The method of claim 3, whereinthe polymer matrix further comprises a nanoparticle.
 5. The method ofclaim 4, wherein the nanoparticle includes ceria.
 6. The method of claim3, wherein the polymer matrix further comprises a glucosidase, a glucosedehydrogenase or a hexokinase.
 7. The method of claim 3, wherein thepolymer matrix further comprises a graphene oxide.
 8. The method ofclaim 3, wherein the polymer matrix further comprises an acrylamide. 9.The method of claim 1, wherein the energy source includes light energy,chemical energy or electrical energy.